The technique of site-directed spin labeling has become a powerful tool for studying protein secondary structure and protein dynamics by EPR spectroscopy. In addition, low frequency CW-EPR techniques for measuring interspin distances of up to 25 A in biradical systems have recently been developed by Shin (Rabenstein, 1995) and Beth (Hustedt, 1997). We are investigating the advantages of using high frequency EPR (HF-EPR) to extract both distance and orientation information from biradical spin systems. We are examining spectra of helical peptides where either the methanthiosulfonate (MTSSL) or TOAC (an unnatural amino acid with a nitroxide as the side chain) spin labels have been incorporated into the peptide sequence at two selected sites. The HF-EPR spectra demonstrate splittings arising from exchange and dipolar couplings between the two nitroxide spin labels. Simulations of the experimental data are currently underway to extract the biradical distance separations and relative orientations. Preliminary simulations indicate that the HF-EPR spectral features are very sensitive to the relative orientation of the two spin labels. This complicates the analysis of the MTSSL biradical peptides where a distribution of orientations is obtained due to the mobility of the tethered nitroxide spin label. In contrast, the more rigid TOAC spin label is more amenable to the extraction of orientation information.